To understand antigen presentation is synonymous with acquiring insight into the biosynthesis of Major Histocompatibility Complex (MHC) products. This process involves not only the synthesis, assembly, and intracellular routing of the MHC products proper, but also requires the proteolysis of antigenic proteins as a source of peptides presented by MHC products. In MHC class II-restricted antigen presentation, proteolysis and sampling of the resultant peptides are essentially confined to the endocytic pathway. The conformational requirements of class II molecules for their proper delivery to the endocytic pathway and their relationship with peptide binding will be explored. Further, the class II associated accessory polypeptide the Invariant chain (Ii), is a major determinant of endosomal targeting of class II molecules, yet the cellular factors that interact with Ii's cytoplasmic tail to accomplish this feat remain elusive. A chemical approach is proposed to generate trimeric versions of Ii's sorting signals, which will be used in immobilized form as an affinity support for the retrieval of interacting proteins, followed by their identification. Little is known with certainty about the identity of the proteases involved in the degradation of specific antigens, nor about the enzymes involved in destruction of Ii, required for liberation of the Class II peptide binding pocket. The final Ii remnant (CLIP) is removed in catalytic fashion and exchanged for antigenic peptide by the H-2M molecule. Using dendritic cell populations from Cathepsin B, D, L and/or S deficient animals, biochemical and antigen presentation experiments are proposed in which the cathespsins' role in generation of peptides and destruction of Ii will be examined, also in the H-2M deficient background to afford greater resolution in the detection of the Ii breakdown intermediates. The dipeptide vinyl 1sulfone, LHVS, is a Cathepsin S-specific inhibitor that will be used as a lead to synthesize a large series of related, radio-labeled vinyl sulfones. In a biochemical approach, these compounds will be used to identify new thiol proteases involved in destruction of Ii and proteolysis of antigen. Proteases are a well-nigh ideal target for pharmacological intervention. The experiments proposed in this application seek to identify the relevant targets in the case of Class II-restricted antigen presentation and should allow the development of new modalities for the intervention in autoimmune disease.